Major Group-B Enterovirus populations deleted in the noncoding 5’ region of genomic RNA modulate activation of the type I interferon pathway in cardiomyocytes and induce myocarditis

Major 5’-terminally deleted (5’TD) RNA forms of group-B coxsackievirus (CVB-5’TD) has been associated with myocarditis in both mice and humans. Although it is known that interferon-β (IFN-β) signaling is critical for an efficient innate immune response against CVB-induced myocarditis, the link between CVB-5’TD RNA forms and type I IFN signaling in cardiomyocytes remains to be explored. In a mouse model of CVB3/28-induced myocarditis, major early-emerging forms of CVB-5’TD RNA have been characterized as replicative viral populations that impair IFN-β production in the heart. Synthetic CVB3/28 RNA forms mimicking each of these major 5’TD virus populations were transfected in mice and have been shown to modulate innate immune responses in the heart and to induce myocarditis in mice. Remarkably, transfection of synthetic viral RNA with deletions in the secondary structures of the 5’-terminal CVB3 RNA domain I, modifying stem-loops “b”, “c” or “d”, were found to impair IFN-β production in human cardiomyocytes. In addition, the activation of innate immune response by Poly(I:C), was found to restore IFN-β production and to reduce the burden of CVB-5’TD RNA-forms in cardiac tissues, thereby reducing the mortality rate of infected mice. Overall, our results indicate that major early-emerging CVB3 populations deleted in the domain I of genomic RNA, in the 5’ noncoding region, modulate the activation of the type I IFN pathway in cardiomyocytes and induce myocarditis in mice. These findings shed new light on the role of replicative CVB-5’TD RNA forms as key pathophysiological factors in CVB-induced human myocarditis.


Introduction
Group-B Enteroviruses (EV-B) are common and ubiquitous human pathogens transmitted through faecal-oral or respiratory routes.Although most EV-B infections remain asymptomatic, group-B coxsackieviruses (CVB) serotypes 1-6 are common causes of severe acute infection in children and young adults, such as myocarditis, pancreatitis or meningitis [1].Acute EV-B infections can evolve toward persistent infections responsible for chronic pathologies such as dilated cardiomyopathy (DCM), type 1 diabetes and inflammatory myopathy [2][3][4].
EV-B are non-enveloped viruses, with single-stranded positive-sense RNA genome of approximately 7,400 nucleotides [1].EV-B RNA genome is flanked on the 5' end by a conserved untranslated region (5'UTR) which is crucial for initiation of viral replication and translation activities [1].A Coxsackievirus-B2 harboring 5'terminally deleted (CVB-5'TD) population ranging from 22 to 36 nucleotides (nt) was found in the heart of a patient who died of fulminant myocarditis [5].Since 2016, our team identified by next generation sequencing (NGS) or rapid amplification of cDNA-end by polymerase chain reaction (RACE-PCR) assays major EV-B populations characterized by 5'UTR deletions ranging from 8 to 50nt (EVB-5'TD), either alone or associated with low proportions of full-length viruses in acute myocarditis or DCM human samples [6][7][8].Similarly, major CVB-5'TD populations have been found in persistent cardiac and pancreatic infections in immunocompetent mouse models [9,10].These EVB-5'TD populations were characterized by a decrease in ratios of positive to negative [(+)/(−)] RNA strands, and low viral protein synthesis activities [6,8,10,11].Taken together these clinical and experimental findings suggest that these replicative EVB-5'TD populations are associated with the development of acute or chronic myocarditis in humans and mouse models [10,12,13].
Reported deletions within the 5'UTR of EV-B forms are known to affect functional secondary-structural elements of the RNA domain I (cloverleaf) [11,14,15].Leveque et al. demonstrated a reduced genomic replicative capacity of these EVB-5'TD RNA forms which was explained by a disturbed binding of cellular factor Poly(rC)-binding protein 2 and viral factor polymerase 3CD on the 5'UTR domain I secondary structure [11].Viral genome maintenance is a key step to develop acute phase of infection and to establish viral RNA persistence in cardiac cells.To achieve viral genome maintenance and long-lasting detection of EV-B in the heart, low-levels replication and translation activities of naturally produced 5'TD viruses might be associated with the capacity to modulate innate immunity activation [16,17].
The innate immune response mediated by type I interferon (IFN) is known to be crucial in the progression of acute viral myocarditis, which may evolve toward either healing or chronic persistent infection [16,18].This crucial role is evidenced by the variety of mechanisms used by RNA(+) viruses to evade or inhibit IFN induction, signalling or effector functions [19][20][21].Type I IFN induction has been shown to protect against EV infection in various models [20,22,23].However, a moderate type I IFN response has been shown to promote persistent viral infections [24,25].It was reported that in a CVB3 mouse model, a reduced type I IFN response resulted in worsening of myocarditis [26].Deletions into the 5'UTR of EV-B populations could modify the RNA secondary-structural elements recognized by innate immune sensors such as RIG-I-like-receptors (RLR, such as Retinoic-inducible gene I, RIG-I or Melanoma differentiation-associated protein 5, MDA5) during EV-B acute infections, resulting in a modulation of viral RNA sensing and type I IFN signalling pathway activation [12,27,28].We recently showed that cardiac and pancreatic acute CVB3/28 infections in immunocompetent mice resulted in a low interferon-β (IFN-β) response [10].Although it is known that IFN-β signaling is critical for an efficient innate immune response against CVB-induced myocarditis, the relationship between CVB-5'TD RNA forms and type I IFN signaling in cardiomyocytes remains to be explored.
In the present report, we first analysed the impact of early emergence of replicative CVB-5'TD RNA forms on cardiac type I IFN levels during a CVB3-induced myocarditis in an immunocompetent DBA/2J mouse mode.Next, synthetic major CVB-5'TD RNA forms were transfected into DBA/2J mice to investigate the direct role of CVB-5'TD RNA forms on the modulation of IFN-β pathway activation and myocarditis pathogenesis.Subsequently, synthetic CVB3/28 RNA forms mimicking early emerging 5'terminal deletions were transfected in cultured human cardiomyocytes to explore the regulatory effect of major viral 5' RNAdomain I deletions on type I IFN pathway induction.Finally, CVB3-infected DBA/2J mice were treated with poly(I:C), an agonist of TLR3 (Toll-like-receptors) and RLR (RIG-I like receptors), to restore an innate immune response.Overall, our experimental results indicate that major CVB-3 populations deleted in the 5' noncoding region in domain I of genomic RNA modulate activation of the type I IFN pathway in cardiomyocytes and induce myocarditis in mice.

Early emergence of 5'terminally deleted CVB populations modulates type I interferon response
To investigate the dynamics of 5'terminally deleted (CVB-5'TD) viral population's emergence in mice, we first quantified total viral RNA loads in cardiac tissues of CVB3/28 infected mice (Fig 1A).Viral RNA loads in the heart exhibited an increase until 2 days post-infection (DPI) (Fig 1A) and then a decrease until 28 DPI but remained detectable with significant mean values of 10 3 cp/μg of cardiac tissues (Fig 1A).Progeny viral production was quantified by classical plaque forming unit assay (PFU), demonstrating a peak at 2 DPI with an absence of detectable infectious particles after 14 DPI, corresponding to the beginning of the viral persistent phase in cardiac tissues (Fig 1B and Table 1).Positive/negative-strand RNA ratio was high at 3 DPI, and low at 14 and 28 DPI, similarly to single-strand/double-strands RNA ratio (Table 1).Encapsidation of viral RNA, demonstrated by a viral entry challenge with or without proteinase K, was maintained at all times post infection (Table 1).Using a RACE-PCR assay followed by a previously validated RNA sequencing strategy by NGS we specifically quantified the proportions of each 5'TD and full-length (CVB-FL) viral populations at different DPI in cardiac tissues.Two major groups of CVB-5'TD populations were characterized: one with

PLOS PATHOGENS
Group-B Enterovirus-5'TD RNA forms impair IFN-β production and induce myocarditis deletions ranging from 8 to 36 nucleotides (nt), with a partial or total loss of stem-loop "b"; and one with deletions ranging from 37 to 50nt, characterized by a partial or total loss of stemloop "c" and a partial loss of stem-loop "d" (Figs 1C and 1D and S1A).These CVB-5'TD populations, significantly major after 2 DPI, were initially associated with CVB-FL that became undetectable after 14 DPI (Fig 1C and 1D).Our results demonstrated an early emergence of two major CVB-5'TD population's groups during acute myocarditis without any detectable CVB-FL forms after 14 DPI, corresponding to the subacute phase of the myocarditis.

PLOS PATHOGENS
Group-B Enterovirus-5'TD RNA forms impair IFN-β production and induce myocarditis I IFN pathway activation in cardiac tissues during the early stage of the CVB3 infection, promoting the development of acute and subsequently chronic myocarditis phases.

In vivo transfection of CVB-5'TD RNA forms induces myocarditis without an IFN-β increase
To better understand the direct link between CVB-5'TD RNA forms and IFN-β modulation, we performed in vivo transfection assays of CVB-FL and 5'TD viral RNA forms.Using Jet-PEI reagent in vivo, by intravenous route, we show that all synthetic RNA forms are detectable in the heart 8 hours after transfection with similar replication activities levels (Fig

Discussion
The emergence of major 5'-terminally deleted RNA forms of group B coxsackievirus (CVB-5'TD) has been associated with myocarditis in both mice and humans [2][3][4]10,13,16,20,32].While type I interferon (IFN) signaling is known to be critical for an efficient innate immune response against EV-B in mouse and human myocarditis, the relationship between CVB-5'TD RNA forms and type I IFN signaling in cardiomyocytes remains to be explored.In a mouse model of CVB3/28-induced myocarditis, we evidenced a major early-emerging replicative CVB-5'TD RNA forms impairing IFN-β production in cardiac tissues.Synthetic CVB3/28 RNA forms mimicking each of these major 5'TD virus populations were transfected alone into mice, modulating innate immune responses in cardiac tissues, and resulting in myocarditis.Deletions in the secondary structures of the 5'terminal CVB3 RNA domain I were found to impair IFN-β production in human cardiomyocytes transfected with synthetic viral RNA.In addition, the poly(I:C)-treatment of infected mice, restored the IFN-β production and reduced the burden of CVB-5'TD RNA -forms in cardiac tissue, thereby decreasing the mortality rate.Overall, our findings demonstrated how natural deletions within the 5'uncoding region affecting the secondary-structural elements of the genomic RNA domain I (cloverleaf structure) modulate type I IFN signaling pathway in cardiomyocytes, promoting the development of acute and chronic CVB3-induced myocarditis of immunocompetent mice.
In the present report, we investigated dynamics of emergence of CVB-5'TD and full-length (CVB-FL) populations in mice cardiac tissues.RNA-dependent RNA polymerase errors and lack of proofreading are likely to explain early generation of CVB-5'TD forms in target cells (Fig 1C) [33].In the heart of CVB3-infected mice, emerging CVB-5'TD populations became major after 48 hours of infection (HPI), in comparison with CVB-FL forms, the minority population thereafter (Fig 1C).These results suggested an evolutionary advantage of CVB-5'TD forms in specific host cell conditions.Recently, we identified that these cardiac early-emerging CVB-5'TD populations associated with CVB-FL forms, were characterized by significant viral genomic replication activities [10,34].The 37-50nt 5'terminal deletions could reduce the binding of specific replication factors to negative-strand RNA [35,36].Loss of such RNA binding might contribute to the significant reduction of positive-strand RNA synthesis and aberrant low (+)/(-) RNA ratios found in CVB3-infected mouse hearts [10,36,37].Consequently, hijacking of viral or cellular host factors of ribonucleoprotein complexes (RNP) by major levels of CVB-5'TD forms could significantly impair genomic replication activities of minor CVB-FL forms explaining why they are undetectable from 14 to 28 DPI in the heart (Fig 1C).Overall, our results suggest that specific host-cell conditions are necessary for emergence and maintenance of CVB-5'TD populations in mouse hearts [10,11,36,38].
These viral RNA are known to be recognized by innate immune sensors such as RIG-I-likereceptors (RLR) resulting in IFN-β signaling pathway activation [15,27].Despite high levels of these replicative viral RNA forms, no strong nor sustained IFN-β pathway activation was observed, revealing a potential immunomodulatory effect of these CVB-5'TD populations (Fig 1E -1G).In comparison, Mengovirus Zn infection induced a strong IFN-β response activation in DBA/2J, demonstrating their ability to produce IFN-β locally in the heart (S1 Fig) .Moreover, others inflammatory cytokines levels were increased during CVB3/28 infection, without correlation with CVB-TD viral loads (S2 Fig) .In order to understand the link between RNA domain I structures and IFN-β signaling pathway induction, we investigated the direct impact of CVB-TD and CVB-FL populations mix in vivo.Inoculation of homogenized mouse hearts from 14 and 28 DPI in mice revealed that CVB-TD populations without CVB-FL forms replicate in hearts and induce myocarditis without an IFN-β production increase (Fig 2B and 2C).Moreover, replicative 8-36nt and 37-50nt CVB-5'TD forms remained detectable in hearts long after the infection and could be persistent forms (S3 Fig) .We then explored how these low-level replicating RNA forms could escape the immune sensors and induce a persistent infection.
Using a strategy based on the transfection of synthetic CVB3/28 RNA mimicking mouse emerging 5'terminal deletions into mouse and human cardiac cells, we studied the direct impact of CVB-FL and 5'TD RNA forms on IFN-β signaling pathway and myocarditis pathogenesis (Figs 3 and 4).Remarkably, in vivo transfection of CVB-5'TD and FL forms revealed that 5'TD21 and 5'TD50 RNA forms can induce inflammatory foci, without IFN-β increase in the heart (Fig 3).This result demonstrates the direct cardiac pathogenicity of these two major CVB-5'TD forms, and their ability to escape innate immune clearance in immunocompetent mouse.In human cardiac cells, we identified stem-loops "b", "c" or "d" of the viral RNA domain I (cloverleaf) as essential viral genomic structures responsible for IFN-β pathway induction (Fig 4).To date, whether the RLR recognition is dependent of RNA sequences or secondary structures remains unknown.We aim to determine the major intra cytoplasmic innate immune sensor (RIG-I or MDA5) responsible for CVB 5'UTR detection using knockout mice or cells for each RLR and to identify viral sequences and RNA secondary-structures that specifically interact with RIG-I or MDA5.
Since we demonstrated that early-emerging CVB-5'TD forms modulate IFN-β signaling pathway activation, we studied the impact of Poly(I:C), a synthetic dsRNA known to induce type I IFN response through TLR3 (Toll-like-receptors 3) and RLR, on CVB3/28-induced myocarditis in DBA/2J mice (Fig 5).We showed that IFN-β signaling pathway activation at the onset of CVB3/28 infection decreased viral replication and translation levels, and the cardiac cells necrosis (Fig 5).IFN-β levels in the heart of infected and treated mice were elevated, in comparison with infected non-treated mice, associated with a significant increase in overall survival (Fig 5).This result highlights the potential therapeutic benefit of immunotherapies in acute or chronic EV-induced myocarditis.
Altogether, these results indicate that modulation of IFN-β by emerging CVB-5'TD forms could be a key step to achieve long-lasting viral genome maintenance in cardiomyocytes, driving the development of CVB3 acute and persistent cardiac infections.IFN-β signaling is known to directly impair picornavirus replication by inducing RNAse L and 2-5(A) synthetase expression [16].The 2-5(A) synthetase/RNAse L pathway, exhibiting ribonuclease activity on double-strand RNA, has been shown to be the major mechanism used by IFN-β to inhibit the replication of picornaviruses [16].Viral populations with 5'terminal deletions in RNA domain I disrupting secondary RNA structures could avoid immune restriction by low IFN-β secretion.Modulation of RLR or TLR3 activation by loss of 5'terminal RNA structures of CVB-5'TD could impair type I IFN pathway activation in infected cells.This mechanism could be a potential way to overcome antiviral immune host response and to lead to the development of sub-acute (14 DPI) and persistent (28 DPI) cardiac infection.
In conclusion, our results indicate that major early-emerging CVB-5'TD forms modulate activation of the type I IFN pathway in cardiomyocytes and induce myocarditis in immunocompetent mice.These findings emphasize the role of replicative CVB-5'TD RNA forms as key pathophysiological players in CVB-induced human myocarditis.Moreover, our results reveal that natural deletions affecting secondary structures of 5'terminal CVB3 RNA domain I can impair innate immune sensing mechanisms in cardiomyocytes during early phase of acute myocarditis.Deciphering the molecular mechanisms leading to the early emergence of CVB-5'TD will enable the development of target immunotherapies to restore an efficient antiviral innate immune response and potentially to achieve a viral clearance in acute and chronic EV-B-induced myocarditis.

Ethic statement
Experiments were performed according to recommendations of the "National Commission of Animal Experiment (CNEA)" and the "National Committee on the Ethic Reflexion of Animal Experiments (CNREEA)".The protocol was approved by the committee of animal experiments of the Reims Champagne-Ardenne (accreditation number 56) and then by Ministry of Advanced Education and research (permits number #37033-2022042914505068).All efforts were made to minimize suffering in accordance with the Guide for the Care and Use of Laboratory Animals of the Direction des Services Ve ´te ´rinaires, the French regulations to which our animal care and protocol adhered.

Cells, virus strain and reagents
HeLa229 cells were grown in minimum essential media (MEM) supplemented by 1% Penicillin-streptomycin (PS) (Gibco, France), 1% L-Glutamine and 10% Fetal Bovin serum (FBS) (ThermoFisher, France).Human primary CardioMyocytes (HCM, ScienCell Research Laboratories) were cultured in cardiac myocyte medium supplemented with 1% PS, 0.01% Cardiac Myocyte Growth Supplement (CMGS) and 5% FBS (ScienCell Research Laboratories) at 37˚C with 5% CO 2 .HCM were passaged twice a week.HCM from ScienCell Research Laboratories were isolated from human adult heart tissues and controlled.Indeed, expansion and long-term cultures result in the selection of non-HCM cells.We used low-passages cells (less than 7 passages), with morphological control of cardiac cells phenotype.CVB3/28 virus strain was gift from Steve Tracy (University of Nebraska Lincoln, USA) [39].CVB3/28 virus is a chimeric

PLOS PATHOGENS
Group-B Enterovirus-5'TD RNA forms impair IFN-β production and induce myocarditis cardiovirulent strain, previously tested in immunocompetent mice and in vitro.Mengovirus WT and Zn strains were gift from Frank Van Kuppeveld (University of Utretch, Netherlands) [29].VP1 antibody raised in rat was a gift from Vesa Hyto ¨nen (University of Tampere, Finland) [40].

Virus production and titration
HeLa229 cells were seeded at 0.8x10 6 cells per well of tissue culture platelet (6 wells) and then incubated at 37˚C overnight.The next day, cell confluence was evaluated at 1,5x10 6 cells per well, based on previous experience, cells were then infected with CVB3/28 at a multiplicity of infection (MOI) of 10 −3 in free-serum MEM.After 48 hours post-infection, the supernatant was collected and then clarified using low-speed centrifugation, virus particles were then measured by plaque-forming-unit assay (PFU).Briefly, HeLa229 cells were infected with CVB3/28, making serial dilutions, for 1h at 37˚C.After viral adsorption, the inoculum was harvested, and cells were overlaid with medium containing 2% agarose and incubated at 37˚C.After 3 days, viral plaques were visualized using bromophenol blue staining.The viral titers were expressed as plaque forming unit per 1 ml (PFU/mL).The deleted 5' UTR viral sequences were obtained as described previously [7].

Infection and transfection of mice
Five-weeks-old male DBA/2J mice, purchased from Charles River laboratory (France), housed under pathogen free condition, were inoculated intraperitoneally with 10 6 PFU/mice (CVB3/28), 10 4 PFU/mice (Mengovirus wild-type or Zn strain) and/or 150μg of Poly(I:C) high molecular weight (HMW) in 150μl of saline; uninfected mice were inoculated with 150μl of saline.Mice were treated with 150μg of Poly(I:C) for two consecutive days after a CVB3/28 intraperitoneal infection (one hour and at day 1post-infection).To determine survival rates upon infection, loss of body weight was monitored daily for 28 days post-infection and mice were euthanized if they had � 20% loss of their initial body weight, according to our protocol.Then mice were anesthetized with Ketamine/Xylazin (42.5/5 mg/kg) and euthanized by cervical dislocation before organs collection at various pre-fixed times points post-infection (1, 2, 3, 7, 14-and 28-days post-infection, DPI).Infected or uninfected mice were sacrificed at the indicated times, a complete autopsy was performed, and hearts were taken and fixed in formaldehyde 4% for histology or homogenized in 600μl of PBS solution with TissueLyser LT (QIAGEN, France).For Fig 3, DBA/2J mice were inoculated intraperitoneally with homogenized hearts collected at 3, 14 or 28 DPI from CVB3-infected mice.To obtain purified supernatants of infected hearts, the heart was first snapfrozen, then homogenized in 600μl of PBS solution with TissueLyser LT.The homogenized heart was centrifugated twice at 1000 rpm during 10 min, followed by the collection of the supernatant.In vivo transfection was performed by intravenous route using In Vivo-jetPEI reagent (Polyplus Transfection, New York, USA) and mice were sacrificed at indicated times post transfection.The transfection mixture consisted of 100 μg of synthetic viral RNA and 5 μl of In Vivo-jetPEI in 5% Glucose solution.Long-RNA (L-RNA) used as control for irrelevant long RNA, was synthetized from the sequence of a pCI Mammalian vector by retrotranscription using TranscriptAid T7 High Yield Transcription kit (Ref K0441-ThermoFisher) (length of RNA~4kb).

Cytokines quantification
The hearts' supernatants were used for: 1) measurement of cytokines by Enzyme-Linked Immunosorbent assay (R&D Systems, France) or ELISA kit for mouse IFN-β (MyBio-Source, USA); 2) virus particles determination as described above.Viral titers were expressed as plaque forming unit per 1 mg of total protein (PFU/mg).Measurement of total protein concentration in each supernatant was performed by the Bicinchoninate Acid Method (Sigma Aldrich, France) according to the manufacturer's instructions.Cytokine protein level was expressed as pg or ng/mg of total protein by calculating the ratio cytokine concentration/total protein concentration.For the quantitative determination of human IFN-β concentrations in cell culture supernatants, ELISA assays using commercially available VeriKine Human IFN Beta ELISA kits (R&D Systems, France) were performed according to the manufacturer's instructions.

Reverse transcription-quantitative PCR using SybrGreen (RT-qPCR)
RNA was extracted from hearts lysates or cultured cells with TRIzol Reagent (Invitrogen, Life Technologies) and reverse transcribed using SuperScript II Reverse Transcriptase (RT) (Invitrogen Life Technologies) following to the manufacturer's instructions.cDNA was subjected to PCR using PowerUp SYBR Green Master Mix (2X) (ThermoFisher Scientific) to detect IFN-β.The primers used for PCR detection as follows: for mouse IFN-β, forward primer 5'-G CCTTTGCCATCCAAGAGATGC-3' and reverse primer 5'-ACACTGTCTGCTGGTGGAG TTC-3'.Primers specific to mouse GAPDH (forward primer 5'-CATCACTGCCACCCAGA AGACTG-3' and reverse primer 5'-ATGCCAGTGAGCTTCCCGTTCAG-3') were used as the internal control.PCR was carried out in StepOnePlus Real-Time PCR Systems (ThermoFisher Scientific) programmed as follows: denaturation step at 94˚C for 15s, annealing step at 63˚C for 10s, and extension step at 72˚C for 15s (40 cycles) for human primers or denaturation step at 95˚C for 15s, annealing/extension step at 65˚C for 1min (40 cycles) for mouse primers, before melting curve.Results were analyzed with ΔΔCT method, where CT is threshold cycle, and normalized to GAPDH mRNA.Data are represented as levels of mRNA relative to that of the mock transfected control samples and are displayed as the means ± SD of results from at least three independent in vitro experiments.

Rapid amplification of cDNA ends-PCR (RACE-PCR) and NGS strategies for CVB-TD detection and characterization
Total nucleic acids extracted from heart tissues were used to generate NGS libraries and RACE-PCR analysis as previously described [6,34].Total RNA extracted from heart tissues were ligated on the 5' extremity with Trp1 RNA adaptator using T4 RNA ligase (Ambion) for 24 hr at 16˚C.Reverse transcription was generated using Superscript II reverse Transcriptase (Life Technologies) and cDNA was performed on ligated RNA using a specific primer (AvcRev) targeting the 5' region of the enteroviral.The cDNAs obtained were then used as a template to perform two PCRs with the KapaTaq polymerase (Clinisciences).These PCR steps amplified the 5' terminal region of ligated viral genomic RNA and added an adapter sequence and barcodes allowing the multiplexing of the samples using primers.The detection and characterization of CVB-TD forms was performed by RACE-PCR assay and then confirmed by NGS.Sequence reads and alignment against the 5'UTR of CVB3/28 were analyzed with a plugging developed by Thermo Fisher Scientifics.Viral populations deleted from less than 8 nucleotides were considered as full-length viral populations [10,34].

Positive and negative-strand RNA ratio
Negative-strand RNA was isolated from total RNA molecules by annealing a biotinylated negative-strand specific primer (E3REV; 5'-GGAACCGACTACTTTGGGTGTCCGTG-3') and binding to streptavidin-labeled magnetic beads (Invitrogen, Life Technologies, Saint-Aubin, France).Purified negative-strand and total viral RNA molecules were quantified with a One-Step real-time RT-PCR assay using serial dilutions of the transcripts for the generation of the standard curves.The positive to negative strand viral RNA ratio was then determined using the following calculation: (total EV RNA-negative strand EV RNA)/negative strand EV RNA.

Single and double-strand RNA ratio
Double-strand EV-B RNA was isolated by digesting single-strand RNA with recombinant bacterial RNase A (ThermoScientific).A mix of 200ng of total RNA with 1μl of RNase and buffer solution of NaCl at 0.3M, were incubated 10min at 25˚C.Double-strand viral RNA copy number was quantified with a One-Step real-time RT-PCR assay using serial dilutions of the transcripts for the generation of the standard curves.The single-strand to double-strand viral RNA ratio was then determined using the following calculation: (total EV RNA-double-strand EV RNA)/double-strand EV RNA.

Replication capacities, progeny and encapsidation assessment
HeLa229 cells were seeded at 1,5x10 5 cells per well of tissue culture platelet (24 wells) and then incubated at 37˚C overnight.The next day, cell confluence was evaluated, based on previous experience, and cells were infected with 300μl of homogenized heart supernatants, for 1h at 37˚C.Encapsidation of these viral RNA forms was assessed by challenging viral entry capacity with or without proteinase K treatment (Merck) which digest all proteins, including viral proteins in the sample.After viral adsorption, the inoculum was collected, and cells were overlaid with free-serum MEM and incubated at 37˚C.After 72 hours, supernatant and cells were harvested separately.Viral loads and titers were measured as described above.

ELISA assays for double-stranded RNA (J2 based) detection
First, total RNA were extracted by Tri-Reagent assay from heart lysat of DBA/2J transfected with FL, TD50, TD21, TD8 and JetPEI (negative control) for 24, 48 and 72 hours.For the detection of double-stranded RNA (dsRNA), ELISA assays using commercially available Double-stranded RNA ELISA kits (J2 based) (Exalpha Biologicals) were performed according to the manufacturer's instructions.Poly(I:C) dsRNA was used as positive control to validate ELISA assay and estimate linear relationship between the amount of dsRNA and the read out for the OD450.

Statistical analysis
Mann-Whitney's test (2 groups) or ANOVA (more than 2 groups) were used for in vivo, in vitro data and ELISA results.Kaplan-Meier test was used to assess the statistical analysis for survival rates.Spearman correlation test was performed.The observed differences were considered significant when P values < 0.05.The number (n) of animal used in the experiment is specified in figure legend.All statistical analyses were performed using GraphPad Prism 8 (Prism).

Fig 1 .
Fig 1.The emergence of 5'terminally deleted (5'TD) CVB populations is associated with a low type I interferon response in the heart.a Viral load levels (RT-qPCR) in the heart of infected mice at 1, 2, 3, 7, 14-and 28-days postinfection (DPI) (n = 4 to 9).b Viral titers (PFU) in the homogenized heart supernatants at 1, 2, 3, 7, 14-and 28 DPI (n = 4 to 9).c.Two-dimensional representation of the EV-B 5'non-coding region RNA sequences showing the position of the CVB3/28 5' terminally deleted forms.d.CVB-5'TD and FL viral loads were assessed using a RACE-PCR method 14 and 28 DPI-infected mice resulted in active viral infections in the heart (Fig 2B).Despite the absence of FL RNA forms in 14 and 28 DPI homogenized-hearts supernatants (Fig 2C), their intra-peritoneal inoculation induced sparse inflammatory foci, VP1 positive detection in cardiac cells and inflammatory cells' infiltrates (Figs 2D andS3).Inoculated CVB3-5'TD populations remained detectable until 42 DPI, indicating a persistent infection (S3A Fig).Mice inoculated with 3-DPI homogenized-hearts experienced a significant increase in IFN-β protein and mRNA levels at 48 HPI (Fig 2Eand 2F).IFN-β production was not or less increased in mice infected with 14 and 28-DPI homogenized hearts, in comparison with those infected with 3-DPI homogenized hearts.Together, these results show that naturally emerging cardiac CVB-5'TD populations can infect and replicate in mice cardiac tissues following an intraperitoneal inoculation, modulating IFN-β production and inducing myocarditis despite the absence of CVB-FL and detectable infectious viral particles.
3A).We show that CVB-FL and CVB-5'TD8 RNA forms transfection induced a significant IFN-β increase in the heart (p = 0.008, p = 0.03 respectively)(Fig 3B).By contrast, CVB-5'TD21 and CVB-5'TD50 RNA forms transfection did not induce any significant variations in IFN-β levels by comparison with the vehicle-transfected-mice (Fig3B).Interestingly, no significant differences were observed in the production of other inflammatory cytokines (CCL2/MCP-1 and TNF-α) after transfection with different 5'TD forms, at different time post-transfection (Fig3C).The induction of inflammatory response by the 5'TD forms could be explained by an active viral genomic replication early after transfection.Using dsRNA ELISA kit, we observed an increase in replicative intermediates viral RNA at 48h post-transfection for TD21 and TD50 (Fig3D).All transfected RNA forms induced inflammatory foci in the heart at 7 DPT, whereas a control Long-RNA did not (Fig 3Eand 3F).Moreover, viral translation activities were assessed by VP1 immunohistochemistry assay in cardiac tissues (Fig3F).CVB-FL and 5'TD-RNA forms transfection resulted in viral protein production in cardiac cells 7 days after transfection.These results demonstrate that CVB-5'TD21 and CVB-5'TD50 RNA forms transfection impair IFNβ production and induce myocarditis in an immunocompetent mice model, by contrast with FL and 5'TD8 RNA transfected forms.